A stilbene synthase from Japanese red pine (Pinus densiflora): Implications for phytoalexin accumulation and down-regulation of flavonoid biosynthesis Atsushi Kodan, Hiroyuki Kuroda*, and Fukumi Sakai Wood Research Institute, Kyoto University, Uji, Kyoto 611-0011, Japan Communicated by Takayoshi Higuchi, Kyoto University, Kyoto, Japan, December 26, 2001 (received for review September 12, 2001) Stilbene synthase (STS) and chalcone synthase (CHS) are plant- specific polyketide synthases that play key roles in the stilbenoid and flavonoid biosyntheses, respectively. We have recently iso- lated from Pinus densiflora three STS cDNAs (PDSTS1, PDSTS2, and PDSTS3) and one CHS cDNA (PDCHSX). We then heterologously expressed these cDNAs in Escherichia coli and characterized their properties. An unusual STS isozyme, PDSTS3, lacks the common C-terminal extension of STS because of a frame-shift mutation and shows the highest pinosylvin-forming activity among the STSs tested. Pinosylvin was shown to be a potent inhibitor of PDCHSX ␮M), which presumably 13 ؍ ␮M) as well as PDSTS2 (Ki 6 ؍ Ki) maintains the balance between the stilbenoid and flavonoid bio- syntheses. PDSTS3 was insensitive to product inhibition. We iden- tified PDSTS3 in the pine seedlings as well as full-length STS. The data provide evidence that PDSTS3 is involved in the potential regulation of the stilbenoid and flavonoid biosynthetic pathways in pine trees. any stress-induced phenylpropanoids are classified as phy- Mtoalexins (1). They have different biological activities and provide an important source of new pharmaceutical and agro- Fig. 1. Pathways for stilbenoid and flavonoid biosyntheses. Stilbene syn- chemical agents. Japanese pines (Pinus densiflora and Pinus thase (STS) and chalcone synthase (CHS), respectively, lead to stilbenoid thunbergii) have been under severe biotic stress over the past 10 and flavonoid biosynthesis from a cinnamoyl-CoA͞p-coumaroyl-CoA with PLANT BIOLOGY decades from pine-wilt diseases, mainly caused by exotic pine- three malonyl-CoAs. PAL, phenylalanine ammonia-lyase; C4H, cinnamate wood nematodes (2). In the genus Pinus, the stilbenoids, pino- 4-hydroxylase. sylvin and its monomethyl ether, are key metabolites that can kill nematodes (3). Stilbenoid phytoalexin formation is controlled by stilbene synthase (STS), which comprises a small gene family in We have recently isolated a structurally unusual STS cDNA, most species examined. The multiplicity of gene family members PDSTS3, from the Japanese red pine, P. densiflora (16). PDSTS3 increases by gene duplications and diverges by base substitution has no common C-terminal extension of STS because of a and insertion͞deletion mutations, leading to functional diver- frame-shift mutation (Fig. 2). Our goal in this article is to gence. Such gene diversity may be selected as a result of biotic compare the enzymological properties of PDSTS3 to those of the stress. Unfortunately, little is known about how general features other two STSs (PDSTS1, PDSTS2) and one CHS (PDCHSX) of enzyme diversity may be of benefit against biotic stress. from P. densiflora. Is PDSTS3 a functional enzyme, and, if so, is STS appears to have evolved from chalcone synthase (CHS) it enzymologically identical to the other STSs; otherwise, does it during land plant evolution (4). STS and CHS belong to a have new functions? Here we report the biochemical properties polyketide synthase (PKS) superfamily and share more than 65% of PDSTS3 as well as PDSTS1, PDSTS2, and PDCHSX, and amino acid homology. Unlike the bacterial PKSs, which are their functional diversity. The biochemical properties of PDSTS3 encoded by large gene clusters (5, 6), both STS and CHS function implicate dynamic regulation of stilbenoid phytoalexin accumu- as unimodular PKSs with a single active site, forming relatively lation and flavonoid biosynthesis. small homodimers (7). STS and CHS use common substrates, three malonyl-CoAs and one cinnamoyl-CoA͞p-coumaroyl- Materials and Methods CoA, forming their products with similar reaction mechanisms Chemicals. [2-14C]Malonyl-CoA (2.04 GBq͞mmol) was pur- (8, 9). STS are classified into either a p-coumaroyl-CoA-specific chased from Amersham Pharmacia. Cinnamoyl-CoA and p- type such as resveratrol synthase (EC 2.3.1.95) or a cinnamoyl- CoA-specific type such as pinosylvin synthase (EC 2.3.1.146) (Fig. 1). For example, the latter type has been reported in Scotch Abbreviations: STS, stilbene synthase; CHS, chalcone synthase; PDSTS1, STS1 from P. den- pine (Pinus sylvestris) (10) and Eastern white pine (Pinus strobus) siflora; PDSTS2, STS2 from P. densiflora; PDSTS3, STS3 from P. densiflora; PDCHSX, CHSX from P. densiflora; PSSTS2, STS2 from P. strobus. (11), whereas the former type has been found in peanut (Arachis Data deposition: The sequences reported in this paper have been deposited in the GenBank hypogaea) (12) and grape (Vitis vinifera) (13). The CHSs from database [accession nos. AB105489 (PDSTS1), AB030139 (PDSTS2), AB030140 (PDSTS3), and many plant sources (nearly 150 CHSs) have been extensively AB015490 (PDCHSX)]. studied, and some of them were defined as naringenin-chalcone *To whom reprint requests should be addressed. E-mail: [email protected]. synthases (EC 2.3.1.74). Recently, the crystal structure of CHS The publication costs of this article were defrayed in part by page charge payment. This from alfalfa was resolved (14). The mechanism of action of other article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. PKSs has also been the subject of considerable attention (15). §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.042698899 PNAS ͉ March 5, 2002 ͉ vol. 99 ͉ no. 5 ͉ 3335–3339 Downloaded by guest on September 28, 2021 STS3, the cells harboring pET32-PDSTS3 were pelleted, har- vested, and resuspended in 20 mM sodium phosphate, pH 7.4͞500 mM NaCl͞60 mM imidazole͞10% (vol/vol) glycerol. After sonication and centrifugation, the supernatant was passed through a Ni2ϩ-nitrilotriacetate (NTA) column, the column was washed with 10 bed volumes of lysis buffer, and the recombinant PDSTS3 was then eluted with lysis buffer containing 250 mM imidazole. The purified recombinant protein was desalted and buffer-exchanged through a prebuffered NICK Spin Column (Sephadex G50 DNA Grade; Amersham Pharmacia). The pro- tein was quantified by using a Coomassie blue protein assay reagent kit (Pierce) with BSA as the standard. STS and CHS Assays. The STS and CHS activities were determined by measuring the conversion of [2-14C]malonyl-CoA into reac- tion products. The reaction mixture contained recombinant STSs or CHS (Ͻ10 pmol), 15 ␮M malonyl-CoA (0.25 kBq), and 20 ␮M cinnamoyl-CoA in 100 ␮l of reaction buffer. The reaction buffer consisted of 20 mM Hepes buffer (pH 7.0), 5 mM EDTA, and 0.3 mM DTT. The mixture was incubated at 30°C for 20 min. The products were extracted twice with ethyl acetate. The Fig. 2. A frame-shift mutation of PDSTS3. (A) Nucleotide deletion in PDSTS3 extracts were evaporated to dryness and redissolved in methanol, cDNA. The two nucleotide deletions are indicated by the hyphens. (B) Align- and the products were separated on Whatman LK6DF silica ment of the deduced amino acid sequences for PDCHSX, PDSTS1, PDSTS2, and TLC plates. The plates were developed with an organic layer of PDSTS3. The frameshift mutation in PDSTS3 changes the amino acid sequence water-saturated diisopropyl ether. The radiograms on an imag- from N293 indicated by the arrow to the newly occurred stop codon indicated ing plate (BAS-IP SR 2025; Fuji) were analyzed by BAS-1800 by the asterisk. (Fuji). The enzyme assays were repeated twice with an appro- priate control assay. coumaroyl-CoA were chemically synthesized (17). Pinosylvin Determination of Inhibition Constant (Ki). For Ki determination, was chemically synthesized by a modified Witig reaction (18). various concentrations (1 ␮Mto500␮M) of either pinosylvin or Resveratrol was purchased from Sigma. Pinocembrin was pur- pinocembrin, dissolved in 50% ethyl cellosolve (4 ␮l), were chased from Indofine Chemical (Somerville, NJ). Naringenin incubated at 30°C with the respective enzyme in the reaction was purchased from Tokyo Kasei (Tokyo). buffer (96 ␮l), which contained different concentrations of cinnamoyl-CoA and 15 ␮M malonyl-CoA. The precise concen- Isolation of STSs and CHS cDNA Clones. Three P. densiflora STS tration range was selected to produce a 20–80% inhibition of the cDNAs (PDSTS1, PDSTS2, and PDSTS3) and chalcone syn- initial activity. Ki was estimated from a double-reciprocal thase cDNA (PDCHSX) were obtained in a previous study (16). Lineweaver–Burk plot. For the PSSTS2 clone from P. strobus, the total RNA was prepared from P. strobus seedlings elicited according to the Preparation of Anti-STS Antibody. The recombinant PDSTS2 an- reported method (11). The first-strand cDNA was synthesized tigen was injected into a rabbit at 2-week intervals. The poly- and PCR-amplified with the primer pairs designed from the clonal antiserum, which had an antibody titer of 10Ϫ6 by ELISA published P. strobus PSSTS2 sequences (11). The product was analysis, was obtained 63 days after the first injection. The identified as PSSTS2 by sequencing both strands (19) on an ABI antiserum was used for immunoprecipitation together with an PRISM 377 DNA sequencer (Applied Biosystems). anti-rabbit IgG-peroxidase conjugate and protein-A agarose (Amersham Pharmacia). Construction of Plasmids and Expression of Recombinant STSs and CHS in Escherichia coli. The STSs and CHS cDNAs were subcloned into Extraction of Crude Enzymes from Pine Seedlings. One gram of the a pET32Xa͞LIC vector (Novagen) and reconfirmed by sequenc- seedlings was ground with liquid nitrogen in a mortar. The ing both strands. The recombinant STSs and CHS are expressed homogenate was extracted with 1 ml of 100 mM Hepes buffer as the fusion proteins with thioredoxin, His-tag, and S-tag at the (pH 7.0) containing 3 mM DTT, 20% (vol/vol) glycerol, and 1% N terminus. E. coli strain Origami B (DE3) was cultured in (wt/vol) BSA, and was squeezed through Miracloth (Calbio- Luria–Bertani medium containing 100 ␮g/ml carbenicillin at chem) and transferred to a new 1.5-ml tube.